FIGURE 4.

IL-1R1 and Nox2 co-localize to lipid rafts both at the plasma membrane and within the redoxosome. MCF-7 cells were transfected with FLAG-IL-1R1 and Nox2 expression plasmids and used for experiments at 48 h post-transfection. A, for an evaluation of IL-1R1 localization to lipid rafts prior to and following stimulation with IL-1β, IL-1R1 and lipid rafts were labeled with anti-FLAG antibody and Alexa Fluor 488-labeled CTX at 4 °C. The cells were then pulsed at 37 °C with or without IL-1β stimulation for 20 min and fixed prior to immunostaining for FLAG. DAPI, 4′,6-diamidino-2-phenylindole. B, the number of IL-1R1- and CTX-positive endosomes per intracellular area was quantified. C, experiments similar to those described for A were performed using an antibody that recognizes an extracellular epitope in Nox2 and Alexa Fluor 488-labeled CTX. The panels show representative examples of Nox2 localization with CTX-positive lipid rafts in the presence and absence of IL-1β stimulation. The arrows mark the lower left corner of the region represented in each enlarged inset. D, the number of Nox2- and CTX-positive endosomes per intracellular area was quantified. For each analysis, a minimum of 24 cells were imaged per condition from a total of three independent experiments. Values represent the mean ± S.D. (n = 3). B and D demonstrate a statistical difference between mock- and IL-1β-stimulated cells as assessed by Student's t test (p < 0.005).